Compositions that have sometimes been used for long-term contraception include those based upon natural or synthetic steroidal hormones to “trick” the female reproductive tract into a “false pregnancy.” These steroidal hormones must be administered repeatedly to prevent completion of the estrous cycle and conception. Steroids have side effects that can be potentially dangerous.
P. Olson et al., “Endocrine Regulation of the Corpus Luteum of the Bitch as a Potential Target for Altering Fertility,” J. Reprod. Fert. Suppl., vol. 39, pp. 27-40 (1989) discusses the luteal phase and its regulation in bitches. The following discussion appears at page 37: “Specific toxins can be linked to an antibody or hormone and carried to a specific target cell (or cells) which is then killed by the toxin. The idea of developing a ‘magic bullet’ has been discussed for decades but is now gaining renewed recognition as a potential, highly selective method for destroying specific tissues while leaving other tissues unharmed. For many years it was impossible to develop large quantities of antibodies which would react specifically with only single antigenic determinants. However, with the advent of monoclonal antibodies, this problem has been largely overcome. Antibodies can be developed to specific hormone receptors (such as the LH receptor) and then coupled to a toxin. All cells with LH receptors should then be destroyed. Although various cell types have not been characterized in dog corpora lutea, destruction of any luteal cell type could potentially result in luteolysis if cell types communicate.” (citations omitted)
P. Olson et al., “New Developments in Small Animal Population Control,” JAVMA, vol. 202, pp. 904-909 (1993) gives an overview of methods for preventing or terminating unwanted pregnancies in small animals. The following discussion appears at page 905: “Tissue-specific cytotoxins—Permanent contraception in females and males might be achieved by administration of a cytotoxin that is linked to gonadotropin-releasing hormone (GnRH) and that selectively destroys gonadotropin-secreting pituitary cells. Similarly, a cytotoxin linked to antibodies against gonadotropin receptors could be targeted to alter gonadal function. Toxins would need to be carefully targeted to specific cells, yet be safe for all other body tissues.” (citation omitted).
T. Janaky et al., “Short Chain Analogs of Luteinizing Hormone-Releasing Hormone Containing Cytotoxic Moieties,” Proc. Natl. Acad. Sci. USA, vol. 89, pp. 10203-10207 (1992) discloses the use of certain hexapeptide and heptapeptide analogs of GnRH as carriers for certain alkylating nitrogen mustards, certain anthraquinone derivatives, antimetabolite, and cisplatin-like platinum complex. The authors reported that several of the compounds exerted some cytotoxic effects on the MCF-7 breast cancer cell line.
D. Fitzgerald et al., “Targeted Toxin Therapy for the Treatment of Cancer,” J. Natl. Cancer Inst., vol.81, pp.1455-1463 (1989), reviewed targeted toxin therapies for cancers, including conjugating toxins such as Pseudomonas exotoxin, diphtheria toxin, and ricin to a cell-binding protein such as a monoclonal antibody or a growth factor. The conjugates are then internalized into cytoplasm, where the toxin disrupts cellular activity.
Conventional targeted toxin therapies have several drawbacks. There is a small window for treatment with a particular targeted toxin (on the order of two weeks) before the recipient's immune system mounts an antibody response to the targeted toxin. These antibodies will neutralize the toxin; or worse, may result in deposition of the toxin in reticuloendothelial tissues (e.g., liver, spleen, lymph nodes, lungs, bone marrow), where they may damage otherwise healthy tissue. Aside from this drawback, the toxin must be internalized by the targeted cell and translocated into the cytoplasm to have effect.
A related approach is to link a monoclonal antibody to an enzyme. This conjugate is directed specifically to a tumor cell surface antigen. A prodrug is then administered to the patient. The prodrug is substantially less toxic than the drug that results from activation of the drug at the tumor site by the conjugated enzyme. The activated drug then selectively attacks tumor cells. See, e.g., D. Kerr et al., “Regressions and Cures of Melanoma Xenografts following Treatment with Monoclonal Antibody β-Lactamase Conjugates in Combination with Anticancer Prodrugs,” Cancer Research, vol.55, pp.3558-3563 (1995); and H. Svensson et al., “In Vitro and In Vivo Activities of a Doxorubicin Prodrug in Combination with Monoclonal Antibody β-Lactamase Conjugates,” Cancer Research, vol. 55, pp.2357-2365 (1995).
S. Sealfon et al., “Molecular mechanisms of ligand interaction with the gonadotropin-releasing hormone receptor,” Endocrine Reviews, vol.18, pp.180-205 (1997) provides a review of research concerning the interaction between GnRH and its receptor.
F. Hu et al., “Theophylline and Melanocyte-Stimulating Hormone Effects on Gamma-Glutamyl Transpeptidase and DOPA Reactions in Cultured Melanoma Cells,” J. Investigative Dermatology, vol. 79, pp. 57-61 (1982) disclosed that theophylline and melanocyte-stimulating hormone (MSH) both enhanced pigmentation in murine melanoma cells, apparently by different mechanisms. J. Murphy et al., “Genetic Construction, Expression, and Melanoma-Selective Cytotoxicity of a Diphtheria Toxin-Related α-Melanocyte-Stimulating Hormone Fusion Peptide,” Proc. Natl. Acad. Sci. USA, vol.83, pp. 8258-8262 (1986) discloses selective activity against melanoma cells in vitro by an MSH-diphtheria toxin conjugate. See also D. Bard, “An Improved Imaging Agent for Malignant Melanoma, Based on [Nle4, D-Phe7]α-Melanocyte Stimulating Hormone,” Nucl. Med. Comm., vol. 16, pp. 860-866 (1995).
W. Siegrist et al., “Homologous and Heterologous Regulation of α-Melanocyte-Stimulating Hormone Receptors in Human and Mouse Melanoma Cell Lines,” Cancer Research, vol. 54, pp. 2604-2610 (1994) reports that it is well-established that human melanoma cells possess specific high affinity receptors for α-MSH. See also J. Tatro et al., “Melanotropin Receptors Demonstrated In Situ in Human Melanoma,” J. Clin. Invest., vol. 85, pp. 1825-1832 (1990).
P. Bacha et al., “Thyrotropin-Releasing Hormone-Diphtheria Toxin-related Polypeptide Conjugates,” J. Biol. Chem., vol. 258, pp. 1565-1570 (1983) discloses conjugates of thyrotropin-releasing hormone (TRH) with two diphtheria toxins; one of these conjugates caused a 50% inhibition of protein synthesis in rat GH3 pituitary cells at 3×10−9 M concentration. See also P. Bacha et al., “Organ-Specific Binding of a Thyrotropin-Releasing Hormone-Diphtheria Toxin Complex after Intravenous Administration to Rats,” Endocrinology, vol. 113, pp. 1072-1076 (1983).
V. Chaudhary, “Activity of a Recombinant Fusion Protein between Transforming Growth Factor Type α and Pseudomonas toxin,” Proc. Natl. Acad. Sci. USA, vol. 84, pp. 4538-4542 (1987) discloses that a fusion protein of a modified Pseudomonas toxin and transforming growth factor type a selectively kills cells expressing epidermal growth factor receptors. See also D. Cawley et al., “Epidermal Growth Factor—Toxin A Chain Conjugates: EGF-Ricin Is a Potent Toxin while EGF-Diphtheria Fragment A is Nontoxic,” Cell, vol. 22, pp. 563-570 (1980).
E. Vitetta et al., “Redesigning Nature's Poisons to Create Anti-Tumor Reagents,” Science, vol. 238, pp. 1098-1104 (1987) reviews the use of immunotoxins against tumors. Uses in preventing graft-versus-host reactions are also mentioned. The authors mentioned that in vivo effectiveness was less than desirable. Difficulties mentioned included accessibility of toxins in circulation to target cells; instability of the linkage of toxin to antibody; rapid clearance of the immunotoxins from circulation by the liver; response by the recipient's immune system to the toxin or to the monoclonal antibody, complicating long-term therapy; possible lack of specificity for neoplastic renewal cells; cross-reactivity with normal cells; heterogeneity of tumor cells; and shedding of surface antigens by tumor cells.
P. Trail et al., “Antigen-specific Activity of Carcinoma-reactive BR64-Doxorubicin Conjugates Evaluated in Vitro and in Human Tumor Xenograft Models,” Cancer Research, vol. 52, pp.5693-5700 (1992) disclose the conjugation of the anticarcinoma antibody BR64 to a doxorubicin derivative, and discuss the antitumor effects of the conjugate.
J. Olson, “Laboratory Evidence for the Hormonal Dependency of Meningiomas,” Human Reproduction, vol. 9, supp. 1, pp. 195-201 (1994) discloses evidence that meningiomas, benign intracranial tumors, possess progesterone receptors.
S. Prigent et al., “The Type 1 (EGFR-Related) Family of Growth Factor Receptors and their Ligands,” Progress in Growth Factor Research, vol. 4, pp. 1-24 (1992) reviews the biology of the epidermal growth factor (EGF), its receptor, and related ligands and receptors (e.g., c-erbB-2, c-erbB-3, TGFα, amphiregulin, heregulin), and their roles in normal cell proliferation and in the pathogenesis of human cancer. See also D. Davies et al., “Targeting the Epidermal Growth Factor Receptor for Therapy of Carcinomas,” Biochem. Pharm., vol. 51, pp. 1101-1110 (1996).
D. Morbeck et al., “A Receptor Binding Site Identified in the Region 81-95 of the β-Subunit of Human Luteinizing Hormone (LH) and chorionic gonadotropin (hCG),” Molecular and Cellular Endocrinology, vol. 97, pp. 173-181 (1993) disclosed a fifteen amino acid region of LH and hCG that acted as a receptor binding site. (LH and hCG are homologous hormones that produce similar effects.)
W. Theunis et al., “Luteinising Hormone, Follicle Stimulating Hormone and Gonadotropin Releasing Hormone Immunoreactivity in Two Insects: Locusta migratoria migratoroides R & F and Sarcophaga bullata (Parker),” Invert. Reprod. and Develop., vol. 16, pp. 111-117 (1989) disclosed that materials immunologically related to LH, FSH, and GnRH were localized in cerebral tissue of Locusta migratona and Sarcophaga bullata. See also P. Verhaert et al., “Substances Resembling Peptides of the Vertebrate Gonadotropin System Occur in the Central Nervous System of Periplaneta americana L.,” Insect Biochem., vol. 16, pp. 191-197 (1986).
U.S. Pat. Nos. 5,378,688; 5,488,036; and 5,492,893 disclose compounds said to be useful in inducing sterility in mammals, and in treating certain sex hormone-related cancers in mammals. The disclosed compounds were generically described as GnRH (or a GnRH analog) conjugated to a toxin. The toxin was preferably linked to the sixth amino acid of the GnRH agonist. The toxin was preferably one with a translocation domain to facilitate uptake into a cell. The inventors noted that conjugation of the GnRH agonist to the toxin “is necessary because, for the most part, the above toxins, by themselves, are not capable of binding with cell membranes in general. That is to say that applicants have found that it is only when a GnRH analog of the type described herein is linked to a toxin of the type noted above does that toxin become capable of binding to cell membranes . . . ” (E.g., U.S. Pat. No.5,488,036, col. 7, lines 46-52.) The toxins specifically mentioned appear all to have been metabolic toxins, for example ricin, abrin, modeccin, various plant-derived ribosome-inhibiting proteins, pokeweed antiviral protein, α-amanitin, diphtheria toxin, pseudomonas exotoxin, shiga toxin, melphalan, methotrexate, nitrogen mustard, doxorubicin, and daunomycin. None of these toxins is believed to be toxic due to direct interaction with the cell membrane. In the in vivo experiments reported, the most effective time course was reported to be weekly injections for 4 weeks. (E.g., U.S. Pat. No. 5,488,036, col. 20, lines 46-47.) Because most of the conjugates cited are relatively large compounds, antigenicity could be a problem when such multiple administrations are used. The GnRH analog was preferably linked to the toxin with one of several specified heterobifunctional reagents. The specifications suggest that considerable effort was expended in conjugating the toxin to the GnRH agonist. The toxins must in general be internalized into the target cells to have effect, and do not act on cell membranes; in addition, at least some of these toxins must be secondarily transported from the membrane-bound vesicle into the cytoplasm to interact with ribosomes, mitochondria, or other cellular components.
M. Kovacs et al., “Recovery of pituitary function after treatment with a targeted cytotoxic analog of luteinizing hormone-releasing hormone,” Proc. Natl. Acad. Sci. USA, vol. 94, pp. 1420-1425 (1997) discloses that a doxorubin analog conjugated to an LH-RH (i.e., GnRH) agonist selectively attacked cells with LH-RH receptors, and that its effect on pituitary cells was reversible. The paper suggests that the conjugate might be used to treat tumors with LH-RH receptors. See also A. Jungwirth et al., “Regression of rat Dunning R-3227-H prostate carcinoma by treatment with targeted cytotoxic analog of luteinizing hormone-releasing hormone AN-207 containing 2-pyrrolinodoxorubicin,” Intl. J. Oncol., vol. 10, pp. 877-884 (1997)
R. Moretti et al., “Luteinizing hormone-releasing hormone agonists interfere with the stimulatory actions of epidermal growth factor in human prostatic cancer cell lines, LNCaP and DU 145,” J. Clin. Endocrin. & Metab., vol. 81, pp. 3930-3937 (1996) discloses that LH-releasing hormone agonists inhibit both androgen-dependent (LNCaP) and androgen-independent (DU 145) human prostatic cancer cell lines, and suggests that the agonists may inhibit proliferation of the tumor cells by interfering with the stimulatory actions of epidermal growth factor.
I. Mezô et al., “Synthesis of GnRH analogs having direct antitumor and low LH-releasing activity,” J. Med. Chem., vol. 40, pp. 3353-3358 (1997) discloses chicken I GnRH agonists and antagonists. Agonist MI-1892 was reported to have low endocrinological activity, but to possess antitumor activity.
A. Nechushtan et al., “Adenocarcinoma cells are targeted by the new GnRH-PE66 chimeric toxin through specific gonadotropin-releasing hormone binding sites,” J. Biol. Chem., vol. 272, pp. 11597-11603 (1997) discloses the use of a Pseudomonas exotoxin coupled to GnRH to kill certain types of cancer cells.
X. Zhu, “Steroid-independent activation of androgen receptor in androgen-independent prostate cancer. A possible role for the MAP kinase signal transduction pathway?” Mol. & Cell. Endocrinol., vol. 134, pp. 9-14 (1997) discloses that androgen receptors in prostate cancer could be activated in the absence of the androgen signal.
G. Emons et al., “Growth-inhibitory actions of analogues of luteinizing hormone releasing hormone on tumor cells,” Trends in Endocrin. Metab., vol. 8, pp. 355-362 (1997) reviews the similarities and differences between GnRH receptors of cancer cells and of normal brain and pituitary cells; and suggests that LHRH analogs interfere with the mitogenic signal transduction of growth-factor receptors and related oncogene products associated with tyrosine kinase activity in a number of malignant human tumors, including breast, ovary, endometrium, and prostate cancers.
D. Tang et al., “Target to Apoptosis: A Hopeful Weapon for Prostate Cancer,” The Prostate, vol. 32, pp. 284-293 (1997) provides a review of research on apoptosis as a route to treat prostate cancers.
A. Goustin et al., “Growth Factors and Cancer,” Cancer Research, vol. 46, pp. 1015-1029 (1986) provides an overview of various growth factors that have been associated with different cancers.
S. Cho et al., “Evidence for autocrine inhibition of gonadotropin-releasing hormone (GnRH) gene transcription by GnRH in hypothalamic GT1-1 neuronal cells,” Mol. Brain Res., vol. 50, pp. 51-58 (1997) discloses that neuroendocrine populations of GnRH neurons have high affinity receptors for GnRH and for GnRH analogs.
S. Sower et al., “Primary structure and biological activity of a third gonadotropin-releasing hormone from lamprey brain,” Endocrinology, vol. 132, pp. 1125-1131 (1993) describes the structure of lamprey III GnRH.
E. Stopa et al., “Immunocytochemical evidence for a lamprey-like gonadotropin-releasing hormone in human brain,” Soc. Neurosci. Abstr., abstract no. 437.8, p. 1577 (1987) discloses that a lamprey-like GnRH III is found in humans.
S. White et al., “Three gonadotropin-releasing hormone genes in one organism suggest novel roles for an ancient peptide,” Proc. Natl. Acad. Sci. USA, vol. 92, pp. 8363-8367 (1995); and J. Powell et al., “Three forms of gonadotropin-releasing hormone characterized from brains of one species,” Proc. Nat. Acad. Sci. USA, vol. 91, pp. 12081-12085 (1994) are examples of papers reporting the typical presence of three forms of GnRH in species of vertebrates.
J. Warnock et al., “Anxiety and mood disorders associated with gonadotropin-releasing hormone agonist therapy,” Psychopharmacology Bull., vol. 33, pp. 311-316 (1997) reports that psychological side effects can accompany chronic treatment with a GnRH agonist.
L. Deligdisch et al., “Pathological changes in gonadotropin releasing hormone agonist analogue treated uterine leiomyomata,” Fertility and Sterility, vol. 67, pp. 837-841 reported the pathological changes associated with treating leiomyomata with a GnRH analog to induce iatrogenic menopause.
J. Fuerst et al., “Effect of active immunization against luteinizing hormone-releasing hormone on the androgen-sensitive Dunning R3327-PAP and Androgen-Independent Dunning R3327-AT2.1 prostate cancer sublines,” Prostate, vol. 32, pp. 77-84 (1997) reported that active immunization of rats with an LHRH-diphtheria toxoid conjugate caused atrophy of the testes, prostate, and androgen-sensitive prostate tumors, with inhibition of the tumors caused by suppression of cell division rather than an increase in cell death; and that the volume increase of androgen-independent prostate tumors was slightly reduced.
C. Mantzoros et al., “Insulin-like growth factor 1 in relation to prostate cancer and benign prostatic hyperplasia,” Br. J. Cancer, vol. 76, pp. 1115-1118 (1997) reported that increased levels of insulin-like growth factor 1 were associated with an increased risk of prostate cancer.
V. Ding, “Sex hormone-binding globulin mediates prostate androgen receptor action via a novel signaling pathway,” Endocrinology, vol. 139, pp. 213-218 (1998) reported that androgen-independent pathways may activate the progression of some prostate cancers.
J. King et al., “Evolution of gonadotropin-releasing hormones,” Trends in Endocrin. Metab., vol. 3, pp. 339-344 (1992) discloses the primary structures of different GnRHs from various vertebrates. See also J. King et al., “Structure of chicken hypothalamic luteinizing hormone-releasing hormone. II. Isolation and characterization,” J. Biol. Chem., vol. 257, pp. 10729-10732 (1982).
N. Mores et al., “Activation of LH receptors expressed in GnRH neurons stimulates cyclic AMP production and inhibits pulsatile neuropeptide release,” Endocrinology, vol. 137, pp. 5731-5734 (1996) discloses that LH acts directly on neuroendocrine neurons in the brain. See also Z. Lei et al., “Signaling and transacting factors in the transcriptional inhibition of gonadotropin releasing hormone gene by human chorionic gonadotropin in immortalized hypothalamic GT1-7 neurons,” Mol. & Cell. Endocrinology, vol. 109, pp. 151-157 (1995).
U.S. Pat. Nos. 5,597,945 and 5,597,946 disclose plants transformed with genes encoding various lytic peptides.